DESCRIPTION (Applicant's Abstract): The cells of the tracheal epithelium play a vital role in pulmonary host defense by maintaining adequate mucociliary clearance. The long-term goal of this project is to elucidate the cellular and molecular mechanisms responsible for regulation of human airway mucociliary clearance. Two major G-protein-coupled signaling systems play important roles in regulating mucociiary clearance: muscarinic receptors (M3) stimulated by acetylcholine (ACh) and purinergic receptors (P2Y) stimulated by ATP or UTP. This application focuses on the regulation of ciliary motility by ACh and ATP/UTP and proposes to extend previous studies using state-of-the-art, single cell optical, biophysical, and molecular techniques to human cells. We have found that both P2Y and M3 receptors have dual, opposing actions on ciliary beating frequency (CBF), actions that seem to be mediated by changes in cytoplasmic Ca2+ concentration ([Ca2+]i). The combination of these two actions serves to give the ciliated cell an extraordinary degree of fine control in regulating CBF responses and may also couple to metabolic activity. In this application, we will examine the role of RGS proteins, a newly described family of regulators of G-protein signaling, and the extent to which they influence [Ca2+]i responses. We will use RGS transfections into cells as tools to manipulate G-protein signaling by measuring the kinetic coupling between changes in Ca2+ and CBF, identifying signaling molecules that mediate such coupling, and advancing our quantitative model of Ca2+ regulation of CBF. The specific aims are: I.) To characterize Ca2+ signaling and CBF in human airway epithelial cells grown at the air-liquid interface. II.) To define the role of RGS proteins in mediating G-protein coupled [Ca2+]i signaling by M3 receptors. III.) To understand coupling between [Ca2+]i and CBF at the molecular level. This project has numerous innovative aspects. We will explore G-protein coupled receptor signaling using transfection of airway cells with plasmids encoding members of the RGS protein family. For these studies, we will use polarized, human airway epithelial cells in which we can measure several physiological responses (Ca2+, CBF) simultaneously at the single cell and single cilium level. This unique capability will advance our knowledge of Ca2+ handling, ciliary motility, and G-protein coupled signaling. Such studies will not only improve our understanding of mucociliary clearance in the airway but also contribute more broadly to our understanding of signal transduction.